EP0353500B1 - Testträger zur analytischen Bestimmung eines Bestandteils einer flüssigen Probe - Google Patents
Testträger zur analytischen Bestimmung eines Bestandteils einer flüssigen Probe Download PDFInfo
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- EP0353500B1 EP0353500B1 EP89112703A EP89112703A EP0353500B1 EP 0353500 B1 EP0353500 B1 EP 0353500B1 EP 89112703 A EP89112703 A EP 89112703A EP 89112703 A EP89112703 A EP 89112703A EP 0353500 B1 EP0353500 B1 EP 0353500B1
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Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
- G01N33/525—Multi-layer analytical elements
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/84—Systems specially adapted for particular applications
- G01N21/8483—Investigating reagent band
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/805—Test papers
Definitions
- the invention relates to a test carrier for the analytical determination of a component of a liquid sample, in particular for the diagnosis of diseases by means of a reaction sequence running on the test carrier.
- the test carrier has several test layers. This includes a base layer on which a sample application area and an evaluation area are located side by side. A liquid transport path extends from the sample application area into the evaluation area.
- the test carrier has a color formation layer in which, based on a color formation reaction with the aid of a color formation reagent system, of which at least one color formation reagent is located in the color formation layer, one for the component to be determined characteristic optically detectable change takes place.
- reagents are embedded in corresponding layers of a solid test carrier which is brought into contact with the sample.
- the sample is usually a body fluid such as blood or urine. However, it can also be a liquid obtained by previous test steps, for example by contacting an eluent with a stool sample.
- the reaction of the liquid sample with the reagents leads to a detectable signal, and the invention relates to cases in which a color change which is characteristic of the component to be determined is produced and which takes place in the color formation layer.
- the color change can be evaluated visually or with the aid of an appropriate device, usually by reflection photometry.
- Different color formation reactions consist of several reaction stages in which several different reagents are involved. These are collectively referred to as the color formation reagent system. At least one color-forming reagent of this system is located in the color-forming layer and takes part in a reaction step that leads to the color change.
- Test carriers are known in many different designs. They are often in the form of a test strip that has one or more test fields on an elongated base layer. For their part, the test fields often consist of several test layers which are of the same size and are arranged one above the other, so that the sample liquid applied to a test field seeps through the test layers perpendicular to the surfaces thereof. In this case, since the liquid transport only takes place transversely to the test layer surfaces (and to the base layer), such test carriers can be referred to as "cross transport test carriers”.
- test carriers with longitudinal transport They generally have an elongated base layer on which a sample application area and an evaluation area can be distinguished. With test carriers of this type, the sample is placed in the sample application area and then transported along a liquid transport path that runs parallel to the base layer into the evaluation area.
- test carriers have considerable advantages over the cross transport test carriers with only test layers arranged one above the other and are particularly well suited for immunological analysis methods.
- the liquid transport is based on capillary action, the liquid transport path being able to be formed both by one or more test layers made of absorbent material, such as paper or nonwoven, and by a gap which is soaked up by capillary action. Test carriers with longitudinal transport are described for example in DE-A 34 45 816, DE-A 36 38 654 and DE-A 36 43 516.
- test carrier It is important for the accuracy and reliable functioning of analysis test carriers that all test layers are sufficiently wetted with sample liquid. On the other hand, to make handling easier, it should not be necessary, if possible, to measure the exact amount of sample liquid before placing it on the test carrier.
- the sample is usually added in excess and waited until the test carrier has completely soaked up. It is usually not possible to check whether or when this actually happened. According to the operating instructions, the user should simply wait for a certain time and then remove the excess sample, for example by wiping or washing off.
- EP-A-256 806 describes a transverse transport test carrier in which the base layer has an opening below the test field. The sample is placed on the test field from the other side. The opening is used to observe the back of the test field by reflection photometry. This should make it possible to determine the point in time at which the test field is completely wetted. However, this procedure is limited to cross transport test carriers. The spread of the liquid over the surface of the test layer can only be checked in the small partial area in which the opening is located, and the possibility of checking is limited to cases in which the sample liquid is colored so that it can be detected immediately.
- the evaluation area above the a surface of the color formation layer facing away from the base layer is arranged a cover layer which is so opaque in the initial state of the test carrier that the color of the color formation layer is not visible.
- the cover layer is in fluid contact with the liquid transport path and the cover layer and the color-forming layer are designed such that the color of the color-forming reagent becomes visible when both layers are wetted by the sample liquid.
- the “dry” state of the test carrier before the sample is placed in is referred to as the initial state.
- fluid contact is a term that has become common in test carrier technology for the fact that two test layers are arranged with respect to one another in such a way that liquid exchange is possible between them, mostly due to the suction effect of the test layers.
- the test layers can be in direct contact with one another or the fluid contact can be indirect, e.g. can be effected via interposed absorbent layers.
- the cover layer consists of an absorbent material.
- an absorbent material for example, paper, a textile material (fabric, fleece) or a porous plastic layer is suitable.
- Any such material has gaps or pores in which the liquid is transported by capillary forces.
- a flat, planar contact should exist between the cover layer and the color formation layer. Both layers can be connected to one another, provided that this does not interfere with the liquid exchange. However, they preferably lie loosely on one another and are pressed against one another in a suitable manner, for example by a hold-down layer.
- What is essential for the present invention is the interaction of the longitudinal transport of the liquid and the visualization of the point in time at which the longitudinal transport is completed.
- the test carrier When the test carrier is brought into contact with the sample in the sample application area, it flows along the test carrier to the detection area. This process is largely independent of the amount of sample applied, as long as it is only sufficient to completely wet all test layers.
- the time when the test carrier is completely filled is now signaled. This not only makes it possible to check that the system is working properly, it is also often used to improve the reproducibility of the liquid dosage.
- the color of the color formation layer becomes visible when the cover layer and the color formation layer are wetted with sample liquid
- the color-forming reagent can penetrate into the cover layer so that its color is visible.
- the color formation reaction preferably takes place predominantly in the cover layer, so that the change in color can be observed well or measured by reflection photometry.
- a color formation layer with a readily soluble color formation reagent can be produced by impregnating a carrier matrix. Since the sample liquid initially spreads laterally in the cover layer (parallel to the layer surface), the problem can arise that a rapidly soluble color-forming reagent accumulates in the progressing liquid front and thus causes an inhomogeneous color formation.
- the liquid transport speed in the cover layer and the start of the color formation reaction in the color formation layer should be coordinated in such a way that the liquid first spreads out in the cover layer and the color formation reaction takes place essentially thereafter.
- the color formation layer is preferably designed such that the color formation reagent is released with a delay. This can be caused by a delayed solubility of the color forming reagent itself.
- the delayed release is preferably achieved by that the color-forming reagent is embedded in a delayed-soluble film layer which is on a support in the form of a film or a fabric.
- the color-forming reagent itself is more rapidly soluble than the film layer in which it is embedded. It is thereby achieved that the color-forming reagent, when released from the signal-forming layer, reacts quickly and homogeneously in the liquid phase.
- the color formation layer preferably contains swellable macromolecules and / or a hydrophobic film former.
- Swellable macromolecules in this sense are also referred to as hydrocolloids. They are macromolecular hydrophilic substances that are soluble in water or at least dispersible and swellable. These include in particular polysaccharides such as exudates (eg gum arabic), seed flours (eg carob bean flour, starch), extracts from plants (e.g. pectins, alginates), microbial polysaccharides (e.g. xanthan gum) and chemically modified polysaccharides (e.g. cellulose derivatives).
- exudates eg gum arabic
- seed flours eg carob bean flour, starch
- extracts from plants e.g. pectins, alginates
- microbial polysaccharides e.g. xanthan gum
- chemically modified polysaccharides e.g. cellulose derivatives
- Certain proteins are also included in this group of substances, e.g. Scleroproteins and their hydrolyzates (e.g. collagen, crotein C), sparingly soluble reserve proteins (e.g. zein, precipitated casein), and hydrophobically drying proteins in correspondingly high concentrations (e.g. serum albumin).
- Scleroproteins and their hydrolyzates e.g. collagen, crotein C
- sparingly soluble reserve proteins e.g. zein, precipitated casein
- hydrophobically drying proteins in correspondingly high concentrations e.g. serum albumin
- Hydrophobic film formers for the purposes of the invention are understood as meaning water-soluble polymers which, when dried from an aqueous solution, form a film which can be detached with delay, in which the film constituents are solidified and bound by the adhesive action of the polymer.
- the dissolution properties of such films can be controlled to a large extent by selection of the polymers.
- polymers include, for example, polyvinyl alcohols, such as those sold under the trademark "Mowiol” by Hoechst AG, Frankfurt, Federal Republic of Germany (FRG), polyethylene oxide, as it is sold under the brand name "Polyox” by Union Carbide Corp., New York, USA , is sold, or acrylic resins, such as those available from Roehm, Darmstadt, FRG under the trademark "Eudragid”.
- a second preferred measure to make the color of the color-forming reagent visible through the cover layer is to produce the cover layer from a material that is in a dry state opaque, but transparent when wet.
- the term "opaque” is to be understood here in such a way that the layer is so largely opaque that the color of the color-forming layer cannot be recognized or can only be recognized very poorly. It is crucial that there is a clear difference in the transparency between the dry and the moist state.
- a plastic membrane ie a fine-pored plastic layer
- a clear difference in the transparency in the moist and dry state is shown by the hydrophilic polyvinylidene difluoride, as described in US Pat. No. 4,618,533.
- the refractive index of the plastic should play an important role here. It can be assumed that materials whose refractive index is close to that of the sample liquid have the property of becoming transparent when wet.
- a cover layer which is transparent in the moist state is used in conjunction with a soluble color formation reagent which passes from the color formation layer into the cover layer.
- the test carrier 1 shown in the figures has a base layer 2 on which the other test layers are attached. In the longitudinal direction, the test carrier can be divided into a sample application area 4 and an evaluation area 6. In the sample application area 4, a conjugate layer 8 and a liquid transport layer 9 are fastened next to one another on the base layer 2 with the aid of hot melt adhesive 10. The layer 8 slightly overlaps the subsequent layer 9 in order to ensure the best possible fluid contact between them. The layers 8 and 9 form a liquid transport path which extends from the sample application and pre-reaction area 4 into the evaluation area 6.
- the sample is applied to the conjugate layer 8, this layer simultaneously serving to carry out a first reaction step.
- the sample application area 4 also serves as a pre-reaction area.
- a color formation layer 11, a cover layer 7 and a hold-down layer 12 can be seen one above the other on the base layer 2.
- the hold-down layer 12 consists of a relatively rigid plastic film. It is attached to the base layer 2 by means of a hot-melt adhesive strip 13 of a correspondingly large layer thickness so that it is parallel to it runs at a distance which corresponds approximately to the total thickness of the color formation layer 11 and cover layer 7.
- the hold-down layer 12 has sufficient rigidity to compress the layers between it and the base layer 2 in such a way that good fluid contact between them is ensured.
- no further absorbent layers are provided on its side facing away from the sample application area 4 in the longitudinal direction of the base layer 2 (ie to the right in FIG. 1 from the color formation layer 11).
- the color formation layer 11 is therefore in fluid contact with the last section of the liquid transport path 8, 9, 7 in the liquid transport direction.
- the color formation layer 11 consists of a carrier film 15 and a delay-soluble film layer 16 thereon, which contains a color formation reagent.
- test carrier shown in the figures is particularly suitable for immunological determinations.
- determinations use highly specific binding reactions between different species, which can be called binding partners.
- Immunological binding partners are, in particular, antibodies on the one hand, and antigens or haptens on the other.
- an antigen AG contained in the sample is to be determined as an analyte
- the following test sequence is typical, for example.
- the sample is applied to the conjugate layer 8.
- the conjugate layer contains a soluble conjugate AKE of an antibody AK that is specifically bindable with the AG with an enzyme E.
- the specific binding reaction gives rise to complexes AG-AKE.
- the AGF is identical to or analogous to the sample antigen, i.e. specifically bindable with the antibody of the AKE contained in the conjugate layer 8.
- the excess free AKE is now fixed to the carrier due to the specific binding reaction with the antigen fixed in layer 9.
- Layer 9 can therefore also be referred to as a “fixing layer”. Only the free AG-AKE complexes reach the evaluation zone 6. As a result, the amount of AG-AKE complexes arriving in the evaluation zone 6 (and thus the amount of the labeling enzyme) corresponds to the amount of the analyte.
- the sample liquid with the AG-AKE complexes continues to flow into the cover layer 7 and fills it completely, essentially before the color formation reaction with the color formation reagent in the layer 11 begins.
- the delayed start of the color formation reaction is achieved in particular by the layer 11 being released with a delay.
- the cover layer 7 is produced in one piece with the fixing layer 9, i.e. both layers consist of a strip of the same layer material. This is preferred but not necessary.
- the cover layer 7 could also be a separate layer which is in fluid contact with the liquid transport path 8, 9 in some way.
- the liquid penetrates perpendicularly to the layer surfaces evenly through the liquid exchange surface 17 shown in dashed lines in FIG. 2 into the color formation layer 11.
- the color formation layer 11 contains a substrate for the enzyme E. Depending on the enzyme concentration, a color change takes place, which is a measure of the concentration of the analyte.
- the visual or technical evaluation of the color change takes place from the side of the cover layer. As long as it is dry, it hides the color of the color formation layer. After the cover layer has filled with liquid and the color-forming layer is at least superficially wetted, however, its color becomes visible. As explained above, this can be achieved in that the color reagent passes from the color formation layer 11 into the liquid phase and continues into the cover layer 7. Additionally or alternatively, the cover layer can consist of a material that becomes translucent in the liquid state.
- the color reagent is colored yellow in the initial state.
- the cover layer is opaque white, so that the evaluation area for the The viewer appears white from the evaluation side.
- the yellow color becomes visible. From this, the observer can recognize that sufficient sample has been applied and that the sample liquid has been transported longitudinally in the test carrier.
- This change in color can also be demonstrated by apparatus. This enables a fully automatic control of the correct function of the test strip.
- the color change can be used to initiate a measurement process.
- This control function is independent of the color of the sample liquid. This is particularly important if largely colorless liquids such as urine or plasma are to be analyzed.
- Thin plastic membranes are particularly suitable as the material for the cover layer and, if appropriate, also for the fixing layer 9 produced in one piece from the same material.
- the material thickness should preferably be less than 1 mm, particularly preferably less than 0.3 mm.
- the following materials are suitable, for example Biodyne BNRG from Pall, Glencove, NY, USA 150 ⁇ m SM 119 from Sartorius, Göttingen, FRG 140 ⁇ m Nitrocellulose from BioRad, Richmond, Ca, USA 130 ⁇ m NC from Schleicher and Schüll, Dassel, FRG 150 ⁇ m
- the following materials are particularly advantageous: they are opaque white when dry and transparent when wet: Durapore and Immobilon, both manufactured by Millipore, Bedford, USA.
- test carrier has been described as an example for the case that an antigen is to be determined.
- An analogous test procedure is also possible for the determination of an antibody, in which case an antigen conjugate in layer 8 and a carrier-fixed analog antibody in layer 9 would have to be used.
- the test carrier according to the invention is particularly suitable for those determinations in which the reaction sequence involves a specific binding reaction between a first binding partner correlated with the concentration of the constituent to be determined (in the AG example) and a marked second binding partner (AKE in the example) to form mobile complexes ( AG-AKE in the example) and a further specific binding reaction between the second binding partner (again AKE in the example) and a third binding partner which is analogous to the first binding partner (AGF in the example).
- the second and third binding partners are each arranged in relation to the direction of flow of the liquid in the test carrier in front of its detection area. This completes the specific binding reaction before the free complexes specific for the analysis reach the detection area.
- the test carrier according to the invention is particularly suitable as a detection unit for a test kit for determining an analyte in the stool, as described in EP-A-291 843.
- This test kit has a sample collection unit in which a liquid containing the analyte is obtained from the stool samples by elution with the aid of an eluent. The sample liquid obtained in this way can advantageously be examined with the test carrier according to the present invention.
- the following example 1 relates to such a test carrier, in which human serum albumin (hSA) is determined as an analyte, which was eluted from a stool sample and is an indicator of the presence of blood in the stool.
- hSA human serum albumin
- a polyester film "Melinex" from ICI, Frankfurt, Germany is used as the base layer.
- the components are bonded using Dynapol S 1358 hot-melt adhesive from Dynamid Nobel, Troisdorf, Germany.
- the cover layer of the test carrier is initially white. Only when the liquid front has reached the covering layer, which takes about 180 seconds, does a color change to yellow occur, which indicates to the user that the sample liquid has completely spread out in the test carrier. Now he can observe the course of the color reaction through the cover layer. In the example, the color changes to red within approximately a further 180 seconds if an amount of human serum albumin above the threshold was contained in the sample.
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Chemical & Material Sciences (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Physics & Mathematics (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE3826057 | 1988-07-30 | ||
DE3826057A DE3826057A1 (de) | 1988-07-30 | 1988-07-30 | Testtraeger zur analytischen bestimmung eines bestandteils einer fluessigen probe |
Publications (3)
Publication Number | Publication Date |
---|---|
EP0353500A2 EP0353500A2 (de) | 1990-02-07 |
EP0353500A3 EP0353500A3 (de) | 1991-01-30 |
EP0353500B1 true EP0353500B1 (de) | 1994-09-21 |
Family
ID=6359987
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP89112703A Expired - Lifetime EP0353500B1 (de) | 1988-07-30 | 1989-07-12 | Testträger zur analytischen Bestimmung eines Bestandteils einer flüssigen Probe |
Country Status (6)
Country | Link |
---|---|
US (1) | US5110550A (es) |
EP (1) | EP0353500B1 (es) |
JP (1) | JPH07104345B2 (es) |
AT (1) | ATE112057T1 (es) |
DE (2) | DE3826057A1 (es) |
ES (1) | ES2061812T3 (es) |
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DE4015378A1 (de) * | 1990-05-14 | 1991-11-21 | Boehringer Mannheim Gmbh | Testtraeger fuer die analytische bestimmung eines bestandteils einer probenfluessigkeit |
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DE4212280A1 (de) * | 1992-04-11 | 1993-10-14 | Boehringer Mannheim Gmbh | Asymmetrisch poröse Membranen |
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CA2167362A1 (en) * | 1995-02-10 | 1996-08-11 | Alexei Dmitri Klimov | Apparatus and method for conducting a binding assay on an absorbent carrier material |
DE19781288B4 (de) * | 1996-11-08 | 2005-09-29 | Amira Medical, Scotts Valley | Opake Reaktionsmatrix zur Analyse von Vollblut |
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US5939252A (en) | 1997-05-09 | 1999-08-17 | Lennon; Donald J. | Detachable-element assay device |
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US9528941B2 (en) | 2012-08-08 | 2016-12-27 | Scanadu Incorporated | Method and apparatus for determining analyte concentration by quantifying and interpreting color information captured in a continuous or periodic manner |
US9285323B2 (en) | 2012-08-08 | 2016-03-15 | Scanadu Incorporated | Quantifying color changes of chemical test pads induced concentrations of biological analytes under different lighting conditions |
CN104969068B (zh) | 2012-08-08 | 2019-06-18 | 思勘度股份有限公司 | 用于在自动校准环境中执行及量化由特定浓度的生物分析物诱发的色彩改变的方法及设备 |
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JP6686278B2 (ja) | 2014-02-28 | 2020-04-22 | 株式会社リコー | 検査装置、検査キット、転写媒体、検査装置の製造方法、及び検査方法 |
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US20160116466A1 (en) | 2014-10-27 | 2016-04-28 | Chembio Diagnostic Systems, Inc. | Rapid Screening Assay for Qualitative Detection of Multiple Febrile Illnesses |
CN108061732B (zh) * | 2018-01-18 | 2023-06-30 | 重庆工商大学 | 一种可穿戴式智能尿检系统及尿检方法 |
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DE3643516A1 (de) * | 1986-12-19 | 1988-06-30 | Boehringer Mannheim Gmbh | Testtraeger fuer die analytische bestimmung von bestandteilen von koerperfluessigkeiten |
US4891313A (en) * | 1988-01-21 | 1990-01-02 | Boehringer Manheim Corporation | Method for determination of a component of a sample |
DE3826056A1 (de) * | 1988-07-30 | 1990-02-01 | Boehringer Mannheim Gmbh | Testtraeger zur analytischen bestimmung eines bestandteils einer fluessigen probe |
-
1988
- 1988-07-30 DE DE3826057A patent/DE3826057A1/de not_active Withdrawn
-
1989
- 1989-07-12 AT AT89112703T patent/ATE112057T1/de not_active IP Right Cessation
- 1989-07-12 DE DE58908389T patent/DE58908389D1/de not_active Expired - Fee Related
- 1989-07-12 EP EP89112703A patent/EP0353500B1/de not_active Expired - Lifetime
- 1989-07-12 ES ES89112703T patent/ES2061812T3/es not_active Expired - Lifetime
- 1989-07-25 US US07/384,726 patent/US5110550A/en not_active Expired - Fee Related
- 1989-07-31 JP JP1197022A patent/JPH07104345B2/ja not_active Expired - Lifetime
Also Published As
Publication number | Publication date |
---|---|
EP0353500A3 (de) | 1991-01-30 |
EP0353500A2 (de) | 1990-02-07 |
JPH07104345B2 (ja) | 1995-11-13 |
US5110550A (en) | 1992-05-05 |
DE58908389D1 (de) | 1994-10-27 |
DE3826057A1 (de) | 1990-02-01 |
ATE112057T1 (de) | 1994-10-15 |
ES2061812T3 (es) | 1994-12-16 |
JPH0278934A (ja) | 1990-03-19 |
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